Astrocytes are the most abundant cell type in the brain. Historically, astrocytes were thought to act primarily as support cells to neurons. However increasing evidence indicates that astrocytes actively participate in brain function in a variety o ways, from serving as neural stem cells in the adult, to regulating synaptic formation and activity. In addition, it has been shown that multiple neurological disorders that have classically been considered to be a result of neuronal dysfunction, such as Rett Syndrome, Fragile X, and amyotrophic lateral sclerosis, have astrocytic components. A more complete understanding of the mechanisms regulating neural circuitry and connectivity is critical to developing therapies for treating neurological disorders, and it is becoming increasingly clear that it will be necessary to incorporate the role of astrocytes. This study proposes to examine the role of astrocytes in mediating the dynamics of dendritic spines in the neocortex of adult mice. Spines are the primary site of postsynaptic communication between neurons, and a subset of spines in the adult brain undergo continued turnover. The continual reorganization of spines facilitates changes in synaptic connectivity, and this plasticity is thought to be the structural basis of learning and memory, as well as recovery after brain injury. Astrocytes actively monitor and respond to synaptic activity, and their processes ensheath dendritic spines, pointing to a direct role for astrocytes in regulating spine dynamics. In this study, a subset of astrocytes expressing the transcription factor, Gli1, will be marked and identified in the cortex, and the ultrastructura relationships between Gli1-expressing astrocytes and spines will be examined. Gli1-expressing astrocytes will then be selectively targeted for ablation, and spines will be repeatedly imaged in vivo using 2 photon laser scanning microscopy to investigate whether basal and activity-dependent spine turnover are impaired following astrocyte ablation. Gli1 expression is stimulated upon exposure to high levels of Sonic hedgehog (Shh) signaling. In order to examine the molecular mechanisms underlying astrocyte-mediated regulation of spine dynamics, Shh activity will be disrupted selectively in astrocytes, and basal and activity-dependent spine dynamics will be examined. This study will provide novel insight into the role of astrocytes in regulating structural plasticity, and further, will reveal the functional significance of Shh signaing in astrocytes. ) PUBLIC HEALTH RELEVANCE: This project proposes to examine the role of astrocytes in mediating the structural plasticity of dendritic spines in the adult neocortex of mice. Understanding the cellular and molecular mechanisms underlying neural circuit connectivity and reorganization are key to restoring or repairing brain function following injury or disease.